bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.156463; this version posted June 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 Novel reference transcriptomes for the

2 Carteriospongia foliascens and

3 Cliona orientalis and associated algal

4 symbiont Gerakladium endoclionum 5 Brian W. Strehlow*1,2,3,4,5,6, Mari-Carmen Pineda5,6, Carly D. Kenkel5,7, Patrick Laffy5, Alan Duckworth5,6,

6 Michael Renton2,8, Peta L. Clode2,3,4, Nicole S Webster5,6,9

7 *corresponding author

8 Author emails, respectively: [email protected], [email protected], [email protected], 9 [email protected], [email protected], [email protected], [email protected], 10 [email protected]

11

12 1 Current address: Department of Biology, Nordcee, University of Southern Denmark, Campusvej 55, 5230 13 Odense, Denmark

14 2School of Biological Sciences, University of Western Australia, 35 Stirling Hwy, Crawley WA 6009, Australia

15 3Centre for Microscopy, Characterisation and Analysis, University of Western Australia, 35 Stirling Hwy, 16 Crawley WA 6009, Australia

17 4Oceans Institute, University of Western Australia, 35 Stirling Hwy, Crawley WA 6009, Australia

18 5Australian Institute of Marine Science, PMB No 3, Townsville MC, Queensland 48106 Western Australian 19 Marine Science Institution, Crawley, WA, Australia

20 6Western Australian Marine Science Institution, 35 Stirling Hwy, Crawley WA 6009, Australia

21 7Department of Biological Sciences, University of Southern California, 3616 Trousdale Parkway, Los Angeles, 22 CA 90089, USA

23 8School of Agriculture and Environment, University of Western Australia, 35 Stirling Hwy, Crawley WA 6009, 24 Australia

25 9Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The University of 26 Queensland, Brisbane, QLD, Australia

27 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.156463; this version posted June 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

28 Abstract

29 Transcriptomes from sponges are important resources for studying the stress responses of these

30 ecologically important filter feeders, the interactions between sponges and their symbionts, and the

31 evolutionary history of metazoans. Here, we generated reference transcriptomes for two common and

32 cosmopolitan Indo-Pacific species: Carteriospongia foliascens and Cliona orientalis. We also created

33 a reference transcriptome for the primary symbiont of C. orientalis – Gerakladium endoclionum. To ensure

34 a full repertoire of transcripts were included, clones of each sponge species were exposed to a range of

35 individual stressors: decreased salinity, elevated temperature, elevated suspended sediment

36 concentrations, sediment deposition and light attenuation. RNA extracted from all treatments was pooled

37 for each species, using equal concentrations from each clone. Sequencing of pooled RNA yielded 409 and

38 418 million raw reads for C. foliascens and C. orientalis holobionts (host and symbionts), respectively. Reads

39 underwent quality trimming before assembly with Trinity. Assemblies were filtered into sponge-specific or,

40 for G. endoclionum, symbiont-specific assemblies. Assemblies for C. foliascens, C. orientalis, and G.

41 endoclionum contained 67,304, 82,895, and 28,670 contigs, respectively. Contigs represented 15,248-

42 37,344 isogroups (~genes) per assembly, and N50s ranged 1,672-4,355 bp. Gene ortholog analysis verified a

43 high level of completeness and quality for sponge-specific transcriptomes, with an average 93% of core

44 EuKaryotic Orthologous Groups (KOGs) and 98% of single-copy metazoan core gene orthologs identified.

45 The G. endoclionum assembly was partial with only 56% of core KOGs and 32% of single-copy eukaryotic

46 core gene orthologs identified. These reference transcriptomes are a valuable resource for future

47 molecular research aimed at assessing sponge stress responses.

48 Keywords

49 Porifera, transcriptome, sponge, Cliona orientalis, Carteriospongia foliascens, Gerakladium endoclionum bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.156463; this version posted June 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

50 Data Description

51 Sponges, phylum Porifera, represent one of the oldest lineages of multicellular [1], hence

52 investigating the transcriptomes of different sponge species can provide insight into the evolution of

53 metazoans and their gene expression profiles. Furthermore, sponges have an uncertain future in the face of

54 global climate change [2,3] as well as local stressors including coastal development, altered hydrological

55 processes, and increased runoff of nutrients, pesticides and sediments [4–7]. Transcriptomic analysis of

56 sponges that have been exposed to different environmental conditions would improve our understanding

57 of the sponge molecular stress response pathways and enhance our ability to effectively manage these

58 ecologically important filter feeders. Although there are approximately 9,000 described sponge species [8],

59 to date only ~35 species have published transcriptomes [9-25] and only ~10 have published genomes

60 [10,16,26–29].

61 In this study, we assembled the transcriptomes of two common and widely distributed Indo-pacific sponge

62 species – Carteriospongia foliascens and Cliona orientalis. Both are emerging model species that have been

63 extensively used to study the physiological and ecological effects of environmental stressors on sponges

64 [30–37]. C. foliascens and C. orientalis are only the second members of their respective orders

65 ( and Clionaida) to have a reference transcriptome sequenced. Whilst both C. foliascens and

66 C. orientalis host diverse populations of bacterial symbionts, e.g. [32], C. orientalis additionally hosts an

67 abundant population of eukaryotic Symbiodiniaceae, Gerakladium endoclionum [38,39], which comprises

68 up to 96% of its algal symbiont community [37]. We used sequences generated from the C. orientalis

69 holobiont, i.e. host and symbiont, to construct a partial reference transcriptome for Gerakladium

70 endoclionum. Matching host and symbiont transcriptomes provide a valuable tool to understand the

71 holobiont response to changing environmental conditions and determine the cause-effect pathways for

72 declining host health with environmental change. These data contribute substantially to available poriferan

73 genetic resources and advance the development of these two sponge species as model systems for field

74 and laboratory studies. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.156463; this version posted June 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

75 Methods

76 Samples and sequencing

77 Samples of C. foliascens and C. orientalis were collected in May 2015 from Fantome Is. (S 18°41.028 E 146°

78 30.706) and Pelorus Is. (S 18°32.903' E 146° 29.172'), respectively, in the central Great Barrier Reef under

79 permits G12/35236.1 and G13/35758.1. As C. orientalis is a bioeroding sponge that encrusts and erodes

80 coral skeletons, five C. orientalis cores (~5 cm in diameter) were collected using an air-drill from a single

81 individual, i.e. cloned, growing on a dead colony of Porites sp. An individual of C. foliascens was cut (cloned)

82 into five pieces as in [32]. Sponges were healed and acclimated under natural light and flow-through

83 seawater for 4 weeks before experiments were performed.

84 In order to capture the full complement of gene expression within the reference transcriptomes, sponges

85 were subjected to five different treatments at the Australian Institute of Marine Science (AIMS) National

86 Sea Simulator: i) decreased salinity, ii) elevated temperature, iii) elevated suspended sediment

87 concentrations (SSCs) and sediment deposition, iv) light attenuation and v) no stress control. Sponge clones

88 were used across all treatments to control for genotype, i.e. one genotype was used per species. Two

89 clones of each species were used for each treatment. In the salinity stress treatment, salinity was

90 decreased from 35 to 22 parts per thousand (ppt) by gradually adding flow-through reverse osmosis (RO)

91 water to the system. Salinity was held constant at 22 ppt for 2 d with flow-through seawater maintained at

92 600 mL min-1. In the heat stress treatment, sponges were exposed to a constant temperature of 32.5˚C for

93 1 d using methods described in [32]. In the sediment treatment, sponges were exposed to elevated SSCs at

94 200 mg L-1 for 1 d as in [40,41], using sediments described therein. In the deposition experiment,

95 sedimentation was approximately 40 mg cm-2, measured using SedPods [42] and sponges were left covered

96 with sediment for 1 d. In the light attenuation treatment, sponges were kept in complete darkness for 2 d.

97 Immediately after each treatment, a sample of sponge tissue (~1 cm3) was flash frozen in liquid nitrogen

98 and stored at -80˚C [43] for RNA extraction and sequencing (RNA-seq). After exposure to the decreased bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.156463; this version posted June 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

99 salinity and darkness treatments, Cliona orientalis was visibly bleached after 2 d, but C. foliascens did not

100 exhibit any colour change. Sponges were not visibly affected, e.g. no bleaching or necrosis, by the sediment

101 exposure or elevated seawater temperature.

102 Approximately 50 mg of each sponge clone was excised and ground using a mortar and pestle. Grinding was

103 performed under a thin layer of liquid nitrogen to limit RNA degradation. All tools were rinsed in ethanol

104 followed by RNase Zap (Sigma-Aldrich, USA) to remove contamination and deactivate RNA degrading

105 enzymes. Total RNA was isolated using the Zymo ZR RNA miniprep kit (Zymo Research, USA), with in-

106 column DNAse digestion, according to the manufacturer’s protocol. Total RNA was subsequently cleaned

107 using the Zymo RNA Clean and Concentrator kit (Zymo Research, USA), following the Manufacturer

108 protocol.

109 Total RNA quality was checked using gel electrophoresis and spectroscopy (NanoDrop 2000c

110 Spectrophotometer, Thermo Fisher Scientific, USA), and quantified using a Quant-iT Ribogreen Assay

111 (Thermo Fisher Scientific, USA). For each sponge species, the RNA from individual treatments was

112 combined in equal amounts (740 ng for C. foliascens and 1,440 ng for C. orientalis) from all sponge clones,

113 to a final total RNA concentration of 3.7 µg in 40 µl of Dnase and Rnase free water (93 ng µl-1) for C.

114 foliascens and 7.2 µg in 55 µl of Dnase and Rnase free water (160 ng µl-1) for C. orientalis. For C. foliascens

115 and C. orientalis respectively, RNA had a ratio of absorbance at 260 nm to 280 nm (A260/A280) of 1.88 and

116 2.02 and an A260/A230 ratio of 1.11 and 1.67. To isolate eukaryotic messenger RNA (mRNA), a TruSeq

117 Stranded mRNA-seq sample prep was performed prior to sequencing. The mRNA was sequenced across

118 two lanes of Illumina HiSeq2500 at the Ramaciotti Centre for Genomics (University of New South Wales,

119 Sydney, Australia) to generate 2 x 100 base pair (bp) paired-end (PE) rapid runs.

120 Transcriptome assembly and annotation

121 Sequencing produced 409 and 418 million raw reads for C. foliascens and C. orientalis, respectively (Table

122 1). Reads were trimmed and assembled using publicly available scripts [44,45] and the protocol detailed in bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.156463; this version posted June 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

123 [46]. Briefly, reads < 50 bp long were removed along with reads containing a homopolymer run of adenine

124 (A) longer than 9 bases using fastx_toolkit [47], and only reads with a PHRED quality score >20 over 80% of

125 the read were retained. TruSeq sequencing adapters and PCR duplicates were also removed [44]. The

126 remaining filtered, high quality reads (32.5, 50.5 million paired reads and 2.9, 8.7 million unpaired reads for

127 C. foliascens and C. orientalis, respectively) underwent de novo assembly into contigs using Trinity v 2.8.5

128 [48]. Data processing and assembly was performed at AIMS in Townsville using a high-performance

129 computer (HPC) and on ABACUS 2.0 at the Danish e-Infrastructure Cooperation (DeiC) National HPC Center.

130 Following assembly, additional quality control was performed to ensure that only target transcripts, i.e.

131 derived from C. foliascens, C. orientalis or G. endoclionum, were included in their respective reference

132 transcriptomes [13,46]. First, contigs less than 400 bp were removed and ribosomal RNA (rRNA),

133 mitochondrial RNA (mtRNA), Symbiodiniaceae, and other non-metazoan (e.g. bacteria) sequences were

134 identified using a series of hierarchical BLAST [49] searches. Transcriptomes were further blasted (BLASTn)

135 against the A. queenslandica rRNA database (SILVA: ACUQ01015651) [50], which was the most complete

136 Poriferan rRNA database. Contigs with a bit-score >45 were removed, i.e. 9 and 10 sequences in C.

137 foliascens and C. orientalis, respectively. This process was repeated using the A. queenslandica

138 mitochondrial genome (NCBI: NC_008944.1 REF), resulting in 61 and 27 sequences being removed from the

139 C. foliascens and C. orientalis assemblies respectively.

140 Remaining contigs were blasted (BLASTx) against the most complete Poriferan (A. queenslandica,

141 aqu2.1_Genes_proteins.fasta) [28,51] and Symbiodinium kawagutii

142 (Symbiodinium_kawagutii.0819.final.gene.pep) [52] predicted proteomes and the NCBI nonredundant (nr)

143 protein database (downloaded September 2019). In order to be included in a sponge-specific assembly,

144 contigs had to return a more significant match (E value ≤ 10-5) to the A. queenslandica proteome compared

145 to blast results from the S. kawagutii proteome and also match a metazoan sequence in the nr database or

146 have no match in the nr database, as described in [13]. Sequences with no match to either proteome were bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.156463; this version posted June 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

147 excluded from the final sponge assemblies [13], a stricter exclusion procedure than used in prior

148 invertebrate transcriptome assemblies [46]. For the C. orientalis holobiont, sequences matching the S.

149 kawagutii proteome more closely than the A. queenslandica proteome (E value ≤ 10-5) and matching to the

150 phylum chromerida in the nr database (or having no match in the nr database) were included in the final G.

151 endoclionum assembly. Although C. foliascens does not contain intracellular Symbiodiniaceae, the

152 decontamination step was also performed in order to remove any potential algal contamination in the

153 sample, resulting in only a few (1,520, 1% of total number of contigs) contaminating sequences being

154 removed.

155 Within each of the three transcriptomes, contigs were assigned to isogroups (~genes) and given gene

156 names and gene ontologies (GO) [53] following the protocol previously described in [44,54]. Briefly, the

157 transcriptomes underwent BLAST pairwise sequence comparison (BLASTx) to the UniProt Knowledgebase

158 (UniprotKB/Swiss-Prot) database [55]. Significant BLASTx results (E value ≤ 10-4) were used by

159 CDS_extractor_v2.pl [56] to extract and identify protein coding sequences. Functional annotations were

160 assigned to isogroups based on orthologous comparisons to the eggNOG 4.5 database [57] using eggnog-

161 mapper [58]. Kyoto Encyclopedia of Genes and Genomes (KEGG) ids were also assigned to isogroups using

162 the KEGG Automatic Annotation server (KAAS) [59]. The guanine-cytosine (GC) content of transcriptomes

163 was calculated using the BBMap package (Joint Genome Institute, USA) [60]. Transcriptome completeness

164 was assessed by Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis [61] on gVolante [62].

165 Assembly evaluation and quality control

166 The holobiont assemblies of C. foliascens and C. orientalis contained 225,126 (N50 = 1,284) and 146,510

167 (N50 = 1,949) contigs greater than 400bp in length (Table 1). After data partitioning, 67,304 and 82,895

168 contigs, for C. foliascens and C. orientalis respectively, were considered the ‘sponge-specific’ transcriptome

169 assemblies. The partitioned G. endoclionum transcriptome isolated from the C. orientalis holobiont

170 comprised 28,670 contigs (Table 1). The C. foliascens, C. orientalis, and G. endoclionum transcriptomes bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.156463; this version posted June 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

171 contained 15,248, 37,344, and 21,566 isogroups, respectively, with mean lengths of 3,024 (N50 = 4,355),

172 1,756 (N50 = 2,369), and 1,375 (N50 = 1,672) bp (Table 1). The number of isogroups identified in the C.

173 foliascens and C. orientalis transcriptomes was comparable to previously published sponge transcriptomes

174 which have reported ~11,000-60,000 expressed genes [15,23,63], although there is considerable variation

175 across species. The G. endoclionum transcriptome, containing 28,670 isogroups was comparable in size to

176 the previously published S. kawagutii genome (36,850 genes) [52] and previously published

177 Symbiodiniaceae transcriptomes, ranging in size from 23,777-26,986 expressed genes [64]. The respective

178 GC content of each assembly was 40.2, 45.5, and 59%, matching reported values for metazoans (35-55%

179 [17,27,65]) and Symbiodiniaceae (45-65% [65]). For C. foliascens and C. orientalis, the percentage of genes

180 assigned a name or GO terms was 64 and 77%, respectively (Table 1), also comparable to other sponge

181 transcriptomes (30-70% [15]) and those of other non-model metazoans (25-62% [14,46]). In comparison to

182 the annotated sponge transcriptomes, only 39% of G. endoclionum isogroups could be assigned function or

183 GO term annotations, however this is consistent with functional annotation of other intracellular

184 Symbiodiniaceae transcriptomes, where between 34-44% of genes were assigned GO terms [64]. The

185 isogroups for C. foliascens, C. orientalis, and G. endoclionum were assigned 3,641, 5,339 and 2,191 unique

186 KEGG annotations respectively.

187 The representative transcriptomes for C. foliascens and C. orientalis are considered largely complete based

188 on BUSCO analysis (92.8% and 94.2% complete, respectively) and the representation of nearly all core

189 eukaryotic Orthologous Groups (KOGs) (97.9% and 98.7% respectively) (Table 1). BUSCO analysis of the

190 transcriptome of G. endoclionum was 32.3% complete and 56% of core KOGs were identified (Table 1). A

191 reduced BUSCO completeness in transcriptomes isolated from intracellular Symbiodiniaceae in corals (33-

192 42%) has been previously reported [66]. The current G. endoclionum transcriptome contained 86% more

193 isogroups than the Symbiodiniaceae transcripts identified within the transcriptome assembly of the closely

194 related sponge holobiont, Cliona varians [17]. C. varians also hosts a congeneric intracellular bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.156463; this version posted June 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

195 photosymbiont, Gerakladium spongiolum [38]. Therefore, the current transcriptome for G. endoclionum

196 was considered useful for future studies, at least for conditions in hospite.

197 Table 1. Assembly statistics for the de novo transcriptomes.

Carteriospongia foliascens Cliona orientalis holobiont holobiont N raw reads (x106) 409 418 N qual filtered: PE, SE (x106) 32.5, 2.9 50.5, 8.7 N contigs holobiont 146,510 225,126 Carteriospongia foliascens Cliona Gerakladium orientalis endoclionum N contigs target species only 67,304 82,895 28,670 Mean GC content target species only 40.2 45.5 59 N genes 15,248 37,344 21,566 Mean contig length (bp) 3,024 1,756 1,375 N50 (bp) 4,355 2,369 1,672 % Annotated 64 77 39 % core KOGs 97.9 98.7 56

BUSCOs N complete (%) 908 (92.8) 921 (94.2) 98 (32.3) N partial (%) 14 (1.48) 16 (1.94) 16 (5.28) N missing (%) 56 (5.73) 38 (3.89) 189 (62.8) 198

199 Re-use potential

200 These reference transcriptomes were assembled to facilitate sponge holobiont research aimed at exploring

201 how both host and symbionts respond to changing environmental conditions. The transcriptomes can be

202 used for studies involving Tag-based RNAseq (TagSeq) [67], a highly accurate [68] and cost-effective

203 sequencing technique for large sample sets. Output files are also formatted for Rank-based Gene Ontology

204 analysis of gene expression data (GO_MWU, [69]), and for Functional Summary and Meta-Analysis of Gene

205 Expression Data (KOGMWU, [70]).

206 Availability of supporting data

207 All data, including raw reads, can be accessed here:

208 https://www.dropbox.com/sh/82ue5l16n4xzxww/AABENUi-Cdbm_z-6x4Gj3qICa?dl=0 . Raw data has also bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.156463; this version posted June 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

209 been deposited on NCBI’s SRA under accession numbers PRJNA639714 and PRJNA639798 for the C.

210 orientalis holobiont and C. foliascens, respectively.

211 Declarations

212 List of abbreviations

213 BUSCO: Benchmarking Universal Single-Copy Orthologs; KEGG: Kyoto Encyclopedia of Genes and Genomes;

214 KOG: EuKaryotic Orthologous Groups; TagSeq: Tag-based RNAseq.

215 Ethics notes and consent for publication

216 Not applicable.

217 Competing interests

218 The authors declare that they have no competing interests.

219 Funding

220 This research was funded by the Western Australian Marine Science Institution (WAMSI) as part of the

221 WAMSI Dredging Science Node, and made possible through investment from Chevron Australia, Woodside

222 Energy Limited, BHP Billiton as environmental offsets and by co-investment from the WAMSI Joint Venture

223 partners. The views expressed herein are those of the authors and not necessarily those of WAMSI. Brian

224 W. Strehlow was supported by a University of Western Australia (UWA) Scholarship for International

225 Research Fees, University International Stipend, and UWA Safety-Net Top-Up Scholarships. Brian W.

226 Strehlow was further supported by the Villum Investigator Grant awarded to Don Canfield (No. 16518). The

227 funders had no role in study design, data collection and analysis, decision to publish, or preparation of the

228 manuscript.

229 Authors' contributions bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.156463; this version posted June 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

230 BWS, MCP, CDK, AD, MR, PC, and NSW conceived and designed the experiments; BWS, MCP, and CDK

231 performed the experiments and laboratory work; BWS performed bioinformatics analyses with extensive

232 help and training from CDK and PL. BWS wrote the first draft. BWS, MCP, CDK, PL, AD, MR, PC, and NSW

233 contributed to revisions of various drafts and approved the final manuscript.

234 Acknowledgements

235 The authors are grateful to E. Botté and G. Millar for their assistance in technical aspects related to

236 computing. They also thank the staff at the AIMS National Sea Simulator for their expertise and assistance

237 in the tank-based experiments. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.156463; this version posted June 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

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